Although transcriptional regulation of gene expression during bacterial growth and stress response has been extensively studied, we have only recently begun to gain insights into how the transcription machinery is spatially organized in the nucleoid in response to growth conditions, and how transcription influences the spatial organization of the nucleoid in the cell. Our current study focuses on co-imaging of the transcription machinery including RNA Pol and transcription factors, DNA and replisomes using superresolution imaging systems. New concepts and/or findings from our research, such as bacterial nucleolus-like structure or organization, nucleolus-like compartmentalization of the transcription factories and spatial segregation of the transcription and replication machineries, have significantly enhanced our understanding of the dynamic landscape of the bacterial chromatin. Our current and future research focuses on testing the following hypotheses: (i) RNA Pol foci are transcription factories or hubs for the expression of growth-promoting genes, which are spatially segregated from transcription silencing territories in the chromosome, and (ii) transcription factories and transcription-silencing territories are reorganized in response to environmental signals. As an example of studying stress responses, we have concentrated on osmotic stress response, which is a conserved process from bacteria to eukaryotes. We co-imaged RNA Pol and DNA, in time-course experiments, in cells after high-salt shock and found that RNA Pol dissociates from the nucleoid during the initial high-salt shock when cytoplasmic K+ increases transiently, followed by RNA Pol reassociation during the later adaptation phase when K+ decreases. In parallel with the dissociation and reassociation of RNA Pol, there are significant changes in nucleoid structure. For current and future studies, we will (i) determine the mechanism(s) underlying the dynamic interaction between RNA Pol and promoter sequences during osmotic stress response in vivo; (ii) study the changes in transcriptome during the stress; and (iii) probe the spatial organization of transcription machinery for the osmotic stress-responsive genes. Together, these studies will provide an integrated view of transcriptional regulation of osmotic stress response in the whole E. coli system. We also extend our basic research to human pathogenic bacteria, mainly H. pylori, which is classified as a Group 1 carcinogen by the World Health Organization. We studied the role of SpoT in cell growth during serum starvation and found that SpoT is important in maintaining cell vitality. SpoT mediates the accumulation of polyphosphate (polyP) during serum starvation, at which time polyP binds to the principal sigma factor, sigma 80, to form highly stable complexes. The target site for the polyP binding is the unique arginine-rich N-terminal region (named the P region) of sigma 80. The putative P region is also found in the major sigma factors of other human pathogens, suggesting a new paradigm for pathogenesis regulation. We will continue to study the interaction between sigma 80 and polyP, the enzyme(s) involved in the metabolism of polyP, and other SpoT-mediated stress responses. Our major effort in the future is to develop an in vitro transcription system, which is an important but challenging task because previous efforts on to do so have not succeeded. We also found that SpoT is important for H. pylori survival in macrophages; we will determine why, and analyze the changes in transcriptomes of both macrophages and H. pylori during infection. Moreover, we will test the hypothesis that genetic and epigenetic variations in H. pylori strains may influence transcription of pathogenic genes. This project was discontinued in December 2018.